Targeting DNA Replication before it Starts

Tumorigenesis and Neoplastic Progression

Targeting DNA Replication before it Starts

Cdc7 as a Therapeutic Target in p53-Mutant Breast Cancers

Sara Rodriguez-Acebes,* Ian Proctor,†

Marco Loddo,* Alex Wollenschlaeger,*Mohammed Rashid,† Mary Falzon,†

A. Toby Prevost,†‡ Richard Sainsbury,§

Kai Stoeber,*† and Gareth H. Williams*†

From the Wolfson Institute for Biomedical Research,* the

Departments of Pathology and UCL Cancer Institute,† and

Surgery,§ University College London, London; and the

Department of Primary Care and Public Health Sciences,‡ Guy’s

Campus, King’s College London, London, United Kingdom

Treatment options for triple-receptor negative (ER�/PR�/Her2�) and Her2-overexpressing (ER�/PR�/Her2�) breast cancers with acquired or de novo resis-tance are limited, and metastatic disease remainsincurable. Targeting of growth signaling networks isoften constrained by pathway redundancy or growth-independent cancer cell cycles. The cell-cycle proteinCdc7 regulates S phase by promoting DNA replication.This essential kinase acts as a convergence point forupstream growth signaling pathways and is thereforean attractive therapeutic target. We show that increasedCdc7 expression during mammary tumorigenesis islinked to Her2-overexpressing and triple-negative sub-types, accelerated cell cycle progression (P < 0.001),arrested tumor differentiation (P < 0.001), genomicinstability (P � 0.019), increasing NPI score (P < 0.001),and reduced disease-free survival (HR � 1.98 [95% CI:1.27–3.10]; P � 0.003), thus implicating its deregulationin the development of aggressive disease. TargetingCdc7 with RNAi, we demonstrate that p53-mutant Her2-overexpressing and triple-negative breast cancer celllines undergo an abortive S phase and apoptotic celldeath due to loss of a p53-dependent Cdc7-inhibitioncheckpoint. In contrast, untransformed breast epithe-lial cells arrest in G1, remain viable, and are able toresume cell proliferation on recovery of Cdc7 kinaseactivity. Thus, Cdc7 appears to represent a potent andhighly specific anticancer target in Her2-overexpress-ing and triple-negative breast cancers. Emerging Cdc7kinase inhibitors may therefore significantly broadenthe therapeutic armamentarium for treatment of the

aggressive p53-mutant breast cancer subtypes identifiedin this study. (Am J Pathol 2010, 177:2034–2045; DOI:

10.2353/ajpath.2010.100421)

Breast cancer is the most frequently diagnosed malig-nancy in women in the Western world and accounts foraround 16% of all cancer death.1 Despite increasingincidence, these mortality figures are decreasing as aresult of widespread screening programs and systemicuse of adjuvant hormonal therapy and chemotherapy.2,3

Moreover, targeted therapies for breast cancer are evolvingrapidly and are broadening available therapeutic options.4,5

Targeting of Her2/neu with trastuzumab has resulted inremarkable reductions in relapse when combined with che-motherapy in Her2-positive breast cancers.6 However, themajority of patients are Her2-negative, and acquired and denovo resistance further limits this type of therapeutic inter-vention. This has led to the targeting of additional compo-nents of growth and survival signaling pathways includingras, raf, Mek, PI3K, and mTOR.7 It is not yet clear howmaximal blockade of vertical signal transduction pathwayswith a combination of receptor and downstream agents willbe tolerated. This approach is further compromised bypathway redundancy and cancer cell cycles becoming in-dependent of upstream growth signaling pathways, so-called autonomous cancer cell cycles.8 In particular, ther-apeutic options for treatment of basal-like cancers areseverely constrained by their estrogen (ER), progesterone(PR), and Her2 triple-receptor negative status. New molec-ularly targeted therapies are therefore urgently required foraggressive breast cancers if further decline in mortality is tobe achieved.

Supported by Cancer Research UK Scientific Program grant C428/A6263(to K.S. and G.H.W.).

S.R.-A. and I.P. contributed equally to this study.

Accepted for publication June 10, 2010.

Supplemental material for this article can be found on http://ajp.amjpathol.org.

Address reprint requests to Dr. Kai Stoeber, Ph.D., Department ofPathology, Rockefeller Building, 21 University Street, London, WC1E 6JJ,UK. E-mail: [email protected].

The American Journal of Pathology, Vol. 177, No. 4, October 2010

Copyright © American Society for Investigative Pathology

DOI: 10.2353/ajpath.2010.100421

2034

An alternative approach to the vertical targeting ofsignal transduction pathways is to direct therapeutic in-terventions downstream at the DNA replication initiationmachinery.8 Cdc7 kinase is a core component of thismachinery and is therefore a potentially attractive targetfor cancer therapy.9 Cdc7 kinase phosphorylates andactivates the Mcm2-7 replicative helicase, an essentialstep for the initiation of DNA synthesis at chromosomalreplication origins.10–12 Cancer cells have been shown toestablish only limited numbers of replication forks underCdc7 rate-limiting conditions, causing fork stalling/col-lapse during an abortive S phase that is followed byapoptotic cell death.13,14 Untransformed human fibro-blasts, on the contrary, appear to avoid lethal S phaseprogression in the presence of low Cdc7 levels by elicit-ing a p53-dependent Cdc7-inhibition checkpoint that ar-rests cells at the G1/S boundary.13 However, it has notyet been established whether this checkpoint is active incell types of epithelial lineage, such as mammary epithe-lial cells. Furthermore, it is currently unclear whether thecell cycle arrest after Cdc7 inhibition is reversible. This isan essential prerequisite in the therapeutic context, as anirreversible cytostatic arrest would cause severe toxicityeffects in self-renewing tissues with high turnover (eg,skin, gut mucosa and bone marrow).

The Mcm2-7 replication initiation factors (MCM) haveemerged as diagnostic and prognostic biomarkers forcancer.8 More recently, we have reported that combinedanalysis of MCM expression and biomarkers of S-G2-Mcell cycle phase progression (eg, geminin, Plk-1, AuroraA, and histone H3) allows in vivo determination of tumorcell cycle kinetics.8 This has lead to the identification ofthree discrete tumor cell cycle phenotypes in breast can-cer: (I) well-differentiated tumors composed predomi-nantly of MCM-negative cells, indicative of an out-of-cycle state; (II) tumors composed of cells with high MCMbut low geminin, Plk-1, Aurora A, and histone H3 phos-phorylated on Ser-10 (H3S10ph) levels, indicative of aG1-delayed/arrested state; and (III) tumors showing highMCM and S-G2-M marker expression, indicative of ac-celerated cell cycle progression (Figure 1).8,15 The ac-celerated cell cycle phenotype had a higher risk of re-lapse when compared with out-of-cycle and G1-delayed/arrested phenotypes (hazard ratio [HR] � 3.90) and wastightly associated with the Her2-overexpressing and tri-ple-receptor negative subtypes.15 Because Cdc7 activityis rate-limiting for entry into S phase, the important ques-tion arises regarding whether overexpression of this es-sential kinase might be linked to breast cancers display-ing the accelerated cell cycle phenotype (phenotype III)and thereby present an attractive target in Her2-overex-pressing and triple-receptor negative cancers. Moreover,a high proportion (40–80%) of these aggressive tumorsubtypes harbor p53 mutations,16–19 suggesting that alarge proportion of these breast cancers are likely torespond to Cdc7 inhibitors due to an impaired Cdc7-inhibition checkpoint.

Here we report the first detailed investigation of Cdc7expression dynamics in breast cancer and investigateCdc7 as a novel therapeutic target in p53mut Her2-over-expressing and triple-receptor negative tumors. Impor-

tantly, we also tested in untransformed breast epithelialcells whether the Cdc7-inhibition checkpoint is revers-ible, a prerequisite for targeting Cdc7 in cancer therapy.

Materials and Methods

Study Cohort

Patients diagnosed with invasive breast cancer between1999 and 2004 were identified retrospectively at Univer-sity College London Hospital (London, UK). Clinical char-acteristics of the patient cohort are presented in Supple-mental Table 1 (at http://ajp.amjpathol.org). Histologicalspecimens were assessed for histological subtype andtumor grade based on Royal College of Pathologistsguidelines and established histological20 and clinical cri-teria.21 Archival patient breast tissue samples were re-trieved from the archives of the UCLH Department ofPathology and included cases spanning histologicalgrades 1–3. The 171 breast cancers were subdividedaccording to their ER, PR, and Her2 receptor status into

Figure 1. Phase-specific distribution of cell-cycle biomarkers in proliferatingcells and out-of-cycle states. Three distinct cell-cycle phenotypes (I, II, andIII) are characterized by the differential expression of cell-cycle biomarkersMcm2, geminin, Plk-1, and histone H3 phosphorylated on Ser-10 (H3S10ph).Prognosis and treatment response can be predicted from the distinct immu-noexpression profile displayed by a tumor.15 DFS indicates disease-freesurvival.

Cdc7 Targeting in p53-Mut Breast Cancer 2035AJP October 2010, Vol. 177, No. 4

three distinct groups: ER�/PR�/Her2� (n � 135), ER�/PR�/Her2� (n � 8), and ER�/PR�/Her2� (n � 28).These subgroups, commonly termed “luminal,” “Her2,”and “triple-negative” cancers, respectively, approximateto subtypes previously defined by gene expression pro-filing.16 Breast cancers were characterized according tocell cycle phenotype as described.15 For each patientthe Nottingham Prognostic Index (NPI) was calculated asdescribed.21 Randomly selected cases of clinically andhistologically normal breast tissue from 21 premeno-pausal women who had undergone reduction mammo-plasty were included in the study. Local research ethicsapproval for the study was obtained from the Joint UCL/UCLH Committees on the Ethics of Human Research.

Antibodies

Rabbit polyclonal antibody (PAb) against human gemininwas generated as described.22 Antibodies were pur-chased from the following suppliers: Mcm2 (BM28, clone46), p21 (SX118), and Rb (G3-245) monoclonal antibod-ies (MAb) from BD Biosciences (Oxford, UK); Mcm2phosphorylated on Ser-53 (Mcm2S53ph) PAb from Be-thyl Laboratories (Montgomery, TX); p53 (Ab-6 DO-1)and poly ADP ribose polymerase (PARP-1) (Ab-2 C-2-10)MAbs from Calbiochem Nottingham, UK); histone�H2A.X and Rb phosphorylated on Ser-807/811(pRbS807/811ph) PAbs from Cell Signaling (Danvers,MA); ER (1D5), Ki-67 (MIB-1) and PR (PgR 636) MAbsfrom Dako (Glostrup, Denmark); Cdc7 MAb from MBLInternational (Woburn, MA); caspase 8 (1-1-37) MAb,histone H3 phosphorylated on Ser-10 (H3S10ph) PAband Plk-1 (35-206) MAb from Millipore (Watford, UK);caspase 3 (CPP32 4-1-18) MAb from Novus Biologicals(Cambridge, UK); caspase 9 (F7) MAb, cyclin A (C-19)PAb, cyclin B (GNS1) MAb, and cyclin E (C-19) PAb fromSanta Cruz Biotechnology (Santa Cruz, CA); and �-actinMAb from Sigma (Dorset, UK).

Immunoexpression Profiling

Paraffin wax-embedded tissue obtained at initial diagno-sis was available for all patients. Tissue blocks werechosen that contained representative tumor sample. Im-munoexpression profiling was performed as described.15

Primary antibodies were applied at the following dilutions:Cdc7 (1:100), ER (1:200), and PR (1:200). Her2 immuno-staining was performed using a HercepTest kit (Dako).Incubation without primary antibody was used as a neg-ative control, and colonic epithelium was used as a pos-itive control. Labeling indices (LI) of the markers in eachtumor were determined as described.15,23–25 To evaluateER and PR expression, the quick (Allred) scoring systemwas used and positivity was defined as a score �2.26

Her2 expression was assessed using the manufacturer’sscoring system.

DNA Image Analysis

For each case, one 40-�m section of paraffin wax-em-bedded tissue obtained from the same block as that

assessed by immunohistochemistry was used to preparenuclei as described.27 Tumor DNA ploidy status wasdetermined using the Fairfield DNA Ploidy System (Fair-field Imaging, Nottingham, UK) as described.15 For sta-tistical analysis, tetraploid and polyploid tumors weregrouped with aneuploid tumors.

Cell Culture, Population Growth Assessment,and Cell Cycle Analysis

BT549 cells (ATCC HTB-122, LGC Standards, Middlesex,UK) and T47D cells (ATCC HTB-133) were cultured inRPMI 1640 medium (Invitrogen, Paisley, UK) supple-mented with 10% fetal bovine serum (FBS, Invitrogen)and 10 �g/ml insulin (Sigma). MDAMB157 (ATCC HTB-24) cells were cultured in DMEM (Invitrogen) plus 15%FBS and 10 �g/ml insulin. MDAMB453 and MDAMB231(ATCC HTB-131 and HTB-26) were cultured in DMEMplus 10% FBS. MCF7 cells (ATCC HTB-22) were grown inMEM (Invitrogen) plus 10% FBS and 10 �g/ml insulin.MCF10A cells (ATCC CRL-10317) were cultured inDMEM/F12 medium (Invitrogen) supplemented with 5%horse serum (Invitrogen), 20 ng/ml human EGF (Prepo-Tech, Rocky Hill, NJ), 0.5 �g/ml hydrocortisone, 100ng/ml cholera toxin, and 10 �g/ml insulin (Sigma). Hu-man Mammary Epithelial Cells (HMEpC) were obtainedfrom ECACC (830-05, Health Protection Agency CultureCollections, Salisbury, UK) and cultured at populationdoublings 10–20 in Mammary Epithelial Cell Growth Me-dium KIT (PromoCell, Heidelberg, Germany). All cellswere cultured at 37°C with 5% CO2. Cell proliferationassessment and flow cytometric cell cycle analysis wereperformed as described.28,29

CldU and IdU Incorporation Assay

Double-labeling experiments with 5-chloro-2�-deoxyuri-dine (CldU) and 5-iodo-2�-deoxyuridine (IdU) (Sigma)were performed as described.30 Primary antibodies usedwere mouse anti–5-bromo-2-deoxyuridine (BrdU) for IdU(B44, BD Biosciences) and rat anti-BrdU for CldU (BU1/75, Abcam, Cambridge, UK). DNA was visualized withDAPI. Confocal fluorescence microscopy of randomfields of nuclei was performed as below and images ofthe DAPI channel (blue), Alexa Fluor 488 channel (green,CldU), and Alexa Fluor 594 channel (red, IdU) were ob-tained. Three hundred to 500 DAPI-stained nuclei (blue)were routinely counted for each treatment, and the per-centage of labeled nuclei was quantified. Images of in-dividual nuclei were acquired at �1000 magnification.

RNA Interference

Small interfering RNA (siRNA) experiments in transformedcell lines were performed as described29 using a specificRNA duplex targeting Cdc7 mRNA (Thermo Scientific, Chi-cago, IL): sense 5�-GCT CAG CAG GAA AGG TGT TTT-3�and antisense 5�-AAC ACC TTT CCT GCT GAG CTT-3�.Nontargeting siRNA (Invitrogen) was used as negative con-

2036 Rodriguez-Acebes et alAJP October 2010, Vol. 177, No. 4

trol for all transformed cells. All transfections were per-formed with Lipofectamine 2000 (Invitrogen) according tothe manufacturer’s directions, and 75 nmol/L (BT549), 100nmol/L (MDAMB157 and MDAMB453), or 10 nmol/L(HMEpC) of CDC7 siRNA was used to achieve efficientknockdown. For double-transfection with CDC7 and p53siRNAs (p53 specific duplex, sense 5�-GGA AGA CUCCAG UGG UAA UUU-3� and antisense 5�- AUU ACC ACUGGA GUC UUC CUU-3� and ON-TARGETplus SMARTpoolTP53 L-003329–00 [Thermo Scientific]), HMEpC andMCF10A cells were first transfected with 10 nmol/L CDC7 orcontrol (Luciferase siRNA, Ambion, Austin, TX) siRNA. After24 hours medium was removed and cells were retrans-fected with control (20 nmol/L), CDC7 (10 nmol/L) pluscontrol (10 nmol/L), or CDC7 (10 nmol/L) plus p53 (9 nmol/Lduplex plus 1 nmol/L SMARTpool) siRNA mixtures. Cellswere harvested at the indicated time points after the secondtransfection. Efficient knockdown was assessed by qRT-PCR and/or Western blot.

Real-Time PCR

Total RNA was isolated from cells with the PureLink Microto-Midi Total RNA Purification System (Invitrogen) according tothe manufacturer’s instructions. Total RNA (40 ng) was re-verse transcribed, and real-time PCR was performed usinga SuperScript III Platinum SYBR Green OneStep qRTPCRkit (Invitrogen) following the manufacturer’s instructions. Re-actions were carried out in an Eppendorf Mastercycler epRealplex Real-Time PCR System (Eppendorf, Cambridge,UK), and results were analyzed with Realplex v1.5 software(Eppendorf). Primer sequences were: CDC7 forward 5�-AACTTGCAGGTGTTAAAAAAG-3� and reverse 5�-TGAAAGTGCCTTCTCCAAT-3�; GAPDH (invariant control)forward 5�-TCAACTACATGGTTTACATGTTC-3� and re-verse 5�-GATCTCGCTCCTGGAAGAT-3�.

BrdU Cell Proliferation Assay

Cells were seeded on glass coverslips and subjected toRNA interference as described above. Before harvest, cellswere pulsed with 10 �mol/L BrdU (Sigma) for 1 hour at37°C. Cells were fixed with 1% paraformaldehyde and per-meabilized with PBS/0.2% Triton X-100. DNA was dena-tured with 2 N HCl for 1 hour and cells were incubated withanti–BrdU-FITC antibody (Alexis Biochemicals, Exeter, UK),50 ng/ml propidium iodide (PI), and 50 ng/ml RNase A (bothfrom Sigma). Each coverslip received a final wash with PBSbefore being mounted in Vectashield (Vector Laboratories,Peterborough, UK) mounting medium. Fluorescence confo-cal microscopy of random fields of nuclei was performed ona Leica TCS SP confocal microscope (Leica, Buckingham-shire, UK). Images were collected, and pictures of the PIchannel (red) and FITC channel (green) were obtainedusing Leica TCS PowerScan software (Leica). The originalmagnification was �200. Three hundred to 500 PI-stainednuclei (red) were routinely counted for each treatment, andthe percentage of nuclei incorporating BrdU (green) wasquantified.

Western Blot Analysis

Whole cell extracts (WCE) were prepared by cell lysis inmodified RIPA buffer (50 mmol/L Tris-Cl pH 7.4, 300 mmol/LNaCl, 0.1% NP40, 1% Triton X-100, 0.5% sodium deoxy-cholate, 0.1% SDS, 5 mmol/L EDTA, 1 mmol/L EGTA, 100mmol/L sodium fluoride and 1 mmol/L sodium orthovana-date) followed by sonication for 10 seconds. Fifty micro-grams of protein was loaded in each lane, separated by4–20% SDS-PAGE, and transferred by semidry electroblot-ting onto Hybond C Extra nitrocellulose membranes (GEHealthcare, Buckinghamshire, UK). Blocking, antibody incu-bations, and washing steps were performed as described.31

TUNEL Assay

Cells were seeded on glass coverslips and subjected toRNA interference as described above. After 96 hourscells were fixed in 1% paraformaldehyde. The TUNELassay was performed using an ApopTag FluoresceinDirect In Situ Apoptosis Detection kit (Millipore) accord-ing to the manufacturer’s instructions. Coverslips werewashed in PBS before being mounted in Vectashield with1.5 �g/ml DAPI. Confocal fluorescence microscopy ofrandom fields of nuclei was performed as describedabove, and images of the DAPI channel (blue) and FITCchannel (green) were obtained. Three hundred to 500DAPI-stained nuclei (blue) were routinely counted foreach treatment, and the percentage of TUNEL-positivenuclei (green) was quantified.

Statistical Analysis

Labeling indices were summarized using median and inter-quartile range. Relationships between Cdc7 expressionand tumor grade, lymph node status, tumor subtype, NPI,DNA ploidy status, and cell cycle phenotype were as-sessed using a combination of nonparametric Jonckheere-Terpstra, Mann–Whitney, and KruskalWallis analysis of vari-ance tests as appropriate (Supplemental Table 2 at http://ajp.amjpathol.org). Analysis of disease-free and overallsurvival was performed using Kaplan–Meier plots (usinghigh and low categories of Cdc7 as above and below themedian value, respectively) and Cox regression (treatingCdc7 expression level as a continuous variable). Hazardratios (with 95% confidence intervals [CI]) for Cdc7 and cellcycle phenotype were estimated separately in univariateanalysis and then combined in multivariate analysis. Pa-tients with incomplete data were excluded from multivariateanalysis. All tests were two-sided and used a significancelevel of 0.05. Analysis was carried out using SPSS 12.0 forWindows (SPSS, Chicago, IL).

Results

Deregulated Cdc7 Expression Is Linked toMammary Epithelial Tumorigenesis

Cdc7 protein levels are tightly down-regulated in qui-escent and differentiated human cells.29 Cdc7 dereg-


ulation leading to elevated levels of this essential ki-nase has been linked to acquisition of the malignant,hyperproliferative phenotype in vitro and in vivo.9

Whether elevated Cdc7 levels are also associated withmammary epithelial tumorigenesis is not known. Toaddress this question, we compared Cdc7 expressionat RNA and protein level in asynchronously proliferat-ing normal (HMEpC), hyperproliferating untransformed(MCF10A), and a panel of malignantly transformed(MCF7, BT549, T47D, MDAMB157, and MDAMB231)breast epithelial cell lines. Compared to primary cells,Cdc7 protein levels were between four- and 10-foldhigher in hyperproliferating and the malignantly trans-formed cell lines as determined by Image J densitom-etry analysis (Figure 2A). Cdc7 mRNA levels were alsoraised in hyperproliferating and transformed cells, butto a lesser degree (Figure 2C). Next, we sought toinvestigate whether elevated Cdc7 levels are also as-sociated with malignant transformation in breast epi-thelial cells in vivo. Epithelial cells of the terminal ductlobular unit appear to reside in a G1 arrested, “li-

censed” (primed) state, indicated by high MCM proteinexpression (median: 33.5%) and the absence ofS-G2-M markers (1%) (Figures 1 and 2B). Consistentwith the finding that mammary epithelial cells withholdfrom progression through S-G2-M phase, Cdc7 ex-pression was very low (median: 0.3%) (Figure 2B). Instriking contrast, progression to the fully developedmalignant phenotype (poorly differentiated, aggressivehigh-grade [grade 3] tumors) is associated with up-regulation of Cdc7 levels (median values: 0.3% versus13.9%, P 0.001) (Figure 2, B and D). This is coupledwith cell cycle progression as indicated by a signifi-cant increase in the proportion of cells expressing theS-G2-M markers geminin (median values: 0.5% versus17.2%, P 0.001), Plk-1 (median values: 0% versus 36%,P 0.001), Aurora A (median values: 0% versus 11.4%,P 0.001) and H3S10ph (median values: 0% versus2.5%, P 0.001) (Figure 2, B and D and data notshown). Taken together, these findings are in keep-ing with the rate-limiting effects of Cdc7 on cellproliferation.

Figure 2. Cdc7 expression in breast epithelial cell lines and tissue. A: Immunoblot analysis of whole cell extracts (WCE) prepared from asynchronouslyproliferating cultures of normal (HMEpC), hyperproliferating (MCF10A), and malignantly transformed (MCF7, BT549, T47D, MDAMB157, and MDAMB231) breastepithelial cells probed with antibodies against Cdc7 and actin (loading control). C: CDC7 mRNA levels relative to GAPDH (invariant control) for each cell line asdetermined by qRT-PCR. B and D: Photomicrographs of tissue sections of representative normal premenopausal breast (B) and high-grade (grade 3) breast cancer(D) immunohistochemically stained with antibodies to the indicated proteins (original magnification, �400). The median labeling index (LI) is shown for eachmarker. Median values and interquartile ranges for the cohort were as follows: Ki-67 40% (25%�69%); Mcm2 93% (70%�100%); Cdc7 14% (9%�22%); geminin17% (11%�25%); Plk-1 14% (9%�20%); and H3S10ph 3% (1%�4%).


Increased Cdc7 Expression Is Associated withArrested Tumor Differentiation and GenomicInstability

Next we examined the relationship between Cdc7 expres-sion and pathological features associated with malignantbehavior and clinical outcome in a cohort of 171 breastcancer cases (Supplemental Table 2 at http://ajp.amjpathol.org). Cdc7 expression showed a strong positive correlationwith tumor grade (P 0.001), increasing markedly with thedegree of arrested differentiation (Supplemental Fig. S1A athttp://ajp.amjpathol.org). Increased Cdc7 expression wasalso associated with acquisition of genomic instability asdefined by DNA ploidy status (P � 0.019; SupplementalFig. S1B at http://ajp.amjpathol.org). In contrast, no signifi-cant association was found between Cdc7 expressionand nodal metastases (P � 0.54; Supplemental Fig.S1C at http://ajp.amjpathol.org). Increased Cdc7 ex-pression was also found to positively correlate withincreasing NPI score (P 0.001) (Supplemental Fig.S2 at http://ajp.amjpathol.org).

Increased Cdc7 Expression Is Associated withAccelerated Cell Cycle Progression, BreastCancer Subtype and Reduced Disease-FreeSurvival

We have previously identified three distinct cell cyclephenotypes in this patient cohort of 171 breast cancers:(I) an “out-of-cycle” state (n � 30); (II) a G1 arrested/delayed state (n � 41); and (III) accelerated S-G2-Mphase progression (n � 100) (Figure 1).15 The acceler-ated cell cycle progression phenotype has a fourfoldhigher risk of relapse when compared with the out-of-cycle and G1-delayed/arrested phenotypes (HR � 3.90[95% CI: 1.81–8.4], P 0.001).15 Extending our reportedfindings, we noted that Cdc7 expression was significantlyincreased in phenotype III tumors compared to pheno-type I and II tumors (median values: 12.3% versus 1.0%and 4.1% respectively, P 0.001) (Figure 3A). The linkbetween high Cdc7 expression levels and acceleratedcell cycle progression is further reflected in the strongpositive correlation between Cdc7 and the S-G2-M phaseprogression markers geminin (Spearman correlation co-efficient � 0.8 [0.74–0.85], P 0.001), Plk-1 (0.74 [0.66–0.8], P 0.001), Aurora A (0.59 [0.48–0.68], P 0.001),and H3S10ph (0.63 [0.53–0.71], P 0.001).

Cdc7 expression was also significantly associated withbreast cancer subtype. In contrast to “luminal” tumors(ER�/PR�/Her2�/�), Cdc7 expression in “Her2” (ER�/PR�/Her2�) and “triple-negative” (ER�/PR�/Her2�) tu-mors was significantly elevated (median values: 5.2%versus 19.6% and 20.6% respectively, P 0.001) (Sup-plemental Fig. S1D at http://ajp.amjpathol.org). Corre-spondingly, the majority of Her2 (91%) and triple-nega-tive tumors (96%) were associated with the activelycycling phenotype III (Figure 3B). Taken together, theseresults establish a strong link between Cdc7 expressionand accelerated cell cycle progression in breast cancer

and show that increased Cdc7 expression is stronglyassociated with both Her2-overexpressing and triple-re-ceptor negative breast cancer subtypes.

We have reported that the accelerated cell cycle pheno-type (III) has a poor prognosis when compared with theout-of-cycle (I) and G1 delayed/arrested (II) phenotypes(HR � 3.90) and is an independent predictor of survivalwhen compared with NPI in multivariate analysis (HR �2.71).15 In keeping with this finding, in the same patientcohort Cdc7 is a predictor of disease-free survival in bothunivariate and multivariate (NPI-adjusted) analyses (HR �1.98 [1.27–3.10], P � 0.003 andHR � 1.80 [1.43–2.28], P �0.025 [per 20% increase in Cdc7 levels] respectively) (Fig-

Figure 3. Relationship between Cdc7 expression, cell cycle phenotype, breastcancer subtype, and disease-free survival. A: The median (solid black line),interquartile range (boxed), and range (enclosed by lines) of Cdc7 expressionacross the breast cancer series are shown according to cell cycle phenotype (seetext and Figure 1). Outlying cases are shown by isolated points. B: Relationshipbetween cell-cycle phenotype and breast cancer subtypes. The panels show theproportion of each breast cancer subtype that display cell-cycle phenotypes I, II,and III. C: Kaplan–Meier curves showing an association between Cdc7 anddisease-free survival (months from diagnosis to death, recurrence, or last follow-up) across the whole breast cancer series.


ure 3C). These data further implicate Cdc7 deregulation inthe development of aggressive disease.

Loss of Cdc7 Function Causes Apoptosis inHer2-Overexpressing and Triple-ReceptorNegative Breast Cancer Cells

The majority of Her2-overexpressing and triple-receptornegative breast tumors harbor p53 mutations.16,18 Becauseloss of functional p53 appears to impair the Cdc7-inhibitioncheckpoint,13 Cdc7 kinase might be a new therapeutictarget in these aggressive tumor subtypes. To test thishypothesis, we have used RNAi against CDC7 in threebreast cancer cell lines (BT549, MDAMB157, andMDAMB45) with molecular characteristics approximating toHer2-overexpressing and triple-negative tumors.32,33

BT549 cells (ER�/PR�/Her2 nonamplified, p53mut) weretransfected with previously characterized CDC7-siRNA.13

Relative to nontargeting control-siRNA (CO), transfectionwith CDC7-siRNA (Cdc7KD) reduced CDC7 mRNA levelsby �90% at 96 hours posttransfection (Figure 4A). Corre-spondingly, in WCE Cdc7 protein was undetectable byWestern blotting from 48 hours to 96 hours posttransfection,with levels increasing again from 120 hours onwards when

the RNAi effect had been washed out (Figure 4A). Consis-tent with efficient Cdc7 depletion at 72 hours posttransfec-tion, Mcm2 phosphorylation at Ser-53, a known phosphositefor Cdc7,34 was abolished (Figure 4, C and D). In addition,CDC7 down-regulation caused a cessation of cell prolifer-ation. Cdc7-depleted cells showed an increase in cell num-bers of just 0.2-fold at 144 hours, compared with increasesof 4.0-fold for untreated (UT) cells and 2.2 fold for control-transfected (CO) cells (Figure 4B). BT549 cells depleted ofCdc7 appeared to enter an abortive S phase as demon-strated by a decrease in both the percentage of BrdU-incorporating cells (23% compared to 48% in CO cells) andthe intensity of fluorescence, which directly correlates withthe amount of BrdU incorporation (Figure 4C). Cdc7-de-pleted cells exhibited an increase in �H2A.X levels, indica-tive of DNA double strand breaks (Figure 4C) and, furthersupporting the notion of an abortive S phase, did notprogress to G2/M phase as demonstrated by the lack ofcyclin B and histone H3 Ser-10 phosphorylation (Figure4C).35 Flow cytometry confirmed a progressive decline inthe G2/M population in Cdc7-depleted cells and revealed aconcomitant increase in the number of cells with less than2C DNA content (Figure 4C). The detection of a sub-G1population of cells, as well as morphological changes such

Figure 4. Cdc7 depletion causes apoptosis in BT549 (triple-receptor negative) breast cancer cells. A: Time course of CDC7 mRNA knockdown (KD) in BT549cells relative to cells transfected with control-siRNA (CO) (upper panel). Immunoblot analysis of untreated (UT), control-siRNA-, and CDC7-siRNA–transfectedBT549 whole cell extracts (WCE) probed with the indicated antibodies (lower panel). B: At the indicated time points, cell number was measured in UT, CO, andCdc7-depleted (Cdc7KD) cell populations. The graph shows fold-increase in cell numbers calculated for each time point relative to the number of cells seeded.C: DNA content of Cdc7KD cells at the indicated times (left panel). At 72 hours posttransfection, BrdU incorporation was assayed. DNA was stained withpropidium iodide (PI). Merged images of the BrdU-FITC (green) and PI (red) channels show the percentage of BrdU-positive cells (center panel). WCE fromUT, CO, and Cdc7KD cells immunoblotted with the indicated antibodies (right panel). D: Cell death and fragmented apoptotic nuclei (inset) were analyzed byphase-contrast microscopy and positive TUNEL staining in Cdc7KD and CO cells (left panel). DAPI was used to stain DNA. Apoptotic cell death was confirmedin Cdc7KD cells by WCE immunoblotting with the indicated antibodies (right panel).


as cell shrinkage and nuclear blebbing and abundantTUNEL staining (Figure 4D) compared to controls suggeststhat failure to elicit the Cdc7-inhibition checkpoint leads toapoptotic cell death in p53mut BT549 cells. As expected,activation of the apoptotic pathway in Cdc7-depletedBT549 cells 96 hours posttransfection was confirmed by amarked decrease in pro-caspase 3, 8, and 9 levels anddetection of the apoptotic cleavage product of poly ADPribose polymerase (PARP-1) (Figure 4D). MDAMB157(ER�/PR�/Her2 nonamplified, p53mut) and MDAMB453(ER�/PR�/Her2 amplified, p53mut) cells responded toCDC7 depletion in a similar manner to BT549 cells byentering an abortive S phase followed by induction of apo-ptosis in a significant proportion of cells (Supplemental Figs.S3 and S4 at http://ajp.amjpathol.org). The in vitro cell linedata suggest that loss of p53 function may either contributeto or be sufficient for abrogation of the Cdc7-inhibitioncheckpoint in mammary epithelium.

Loss of Functional p53 Disables theCdc7-Inhibition Checkpoint in UntransformedBreast Epithelial Cells

To further investigate the link between p53 function andthe Cdc7-inhibition checkpoint response, we first askedwhether the checkpoint is active in untransformed mam-mary epithelial cells. To address this question, HMEpCand immortalized nontumorigenic MCF10A cells (p53wt)were first transfected with CDC7-siRNA. Relative to con-trol-siRNA (CO) at 72 hours posttransfection, CDC7-siRNA (Cdc7KD) reduced CDC7 mRNA levels in HMEpCand MCF10A cells by 90% and 95%, respectively (datanot shown). At this time point, Cdc7 protein levels wereundetectable in WCE prepared from MCF10A cells andwere found to be markedly reduced in HMEpC cells(Figure 5A and Supplemental Fig. S5A at http://ajp.amjpathol.org). In both lines, Cdc7 levels eventually re-covered to a detectable level after washing out of theRNAi effect (at 96 hours in MCF10A cells and 144 hoursin HMEpC, reflecting the shorter population doubling timeof the immortalized line). CDC7 down-regulation pre-vented an increase in cell numbers up to 96 hours post-transfection in both cell lines (Figure 5B and Supplemen-tal Fig. S5B at http://ajp.amjpathol.org). Cell proliferationresumed on recovery of Cdc7 levels at later time points,indicating that untransformed mammary epithelial cellsretain the ability to re-enter the cell division cycle afterCdc7 levels are restored. Flow cytometry revealed thatthe majority of Cdc7-depleted HMEpC and MCF10A cellsaccumulated with G1 DNA content consistent with eithera G1 or early S-phase arrest, while only a small fractionshowed G2/M content (Figure 5C and Supplemental Fig.S5C at http://ajp.amjpathol.org). In keeping with the cellcycle profiles, the percentage of cells incorporating BrdUfell from 26% and 34% in control-transfected cells to 6%and 9% in Cdc7-depleted HMEpC and MCF10A cells(Figure 5C; and Supplemental Fig. S5D at http://ajp.amjpathol.org), indicating that the majority of cells hadfailed to synthesize DNA. Western blotting of WCE pre-pared from Cdc7-depleted HMEpC and MCF10A cells 72

hours posttransfection showed a marked increase in cy-clin E levels, while levels of the S phase cyclin A and themitotic cyclin B were reduced below the detection limit(Figure 5C and Supplemental Fig. S5E at http://ajp.amjpathol.org). Both Cdc7-depleted cell lines alsoshowed loss of Rb phosphorylation at Ser-807/811,thought to be either Cdk4 or Cdk2 phosphorylationsites,36,37 p53 stabilization and induction of p21 expres-sion (Figure 5C and Supplemental Fig. S5E at http://ajp.amjpathol.org). Only a minute fraction of Cdc7-de-pleted HMEpC and MCF10A cells with less than 2C (subG1) DNA content was detected by flow cytometry (Figure5C and Supplemental Fig. S5C at http://ajp.amjpathol.org). Moreover, compared to control-transfected cells,neither an increase in cells with morphological features ofapoptosis and positive TUNEL staining of nuclei (Figure5D and Supplemental Fig. S5F at http://ajp.amjpathol.org)nor cleavage of pro-caspases 3 and 9 was observed ineither cell line (Figure 5D and Supplemental Fig. S5E athttp://ajp.amjpathol.org), indicating that arrested cells didnot activate the cell death effector machinery. Taken to-gether, the cell cycle phase distribution and cyclin profiles,low CDK activity evident from hypophosphorylated pRb,and the loss of BrdU incorporation support the notion that, inline with other untransformed cell types, breast epithelialcells operate a Cdc7-inhibition checkpoint that arrests thecell cycle at the G1-S boundary in response to low Cdc7levels.

To test the supposition that loss of p53 function issufficient to abrogate the Cdc7-inhibition checkpoint inmammary epithelial cells, we used RNAi to down-regu-late p53 in HMEpC cells arrested by Cdc7 depletion.Western blot analysis of WCE prepared from cotrans-fected cells 72 hours posttransfection shows down-reg-ulation of p53 to below baseline levels found in controlcells, which in turn resulted in p21 down-regulation (Fig-ure 5, A and C). Flow cytometry analysis of doubly de-pleted Cdc7/p53 cells shows an increase in the numberof cells with S phase and G2/M DNA content 72 hoursposttransfection. The number of cells with less than 2CDNA content progressively increased between 72 and144 hours, coinciding with a sharp decline in the G2/Mpopulation (Figure 5C). Notably, in doubly depletedCdc7/p53 cells Rb phosphorylation at Ser-807/811 in-creased, indicating recovery of CDK activity which is inkeeping with low p21 levels (Figure 5C). The notion thatcells lacking p53 function fail to maintain the Cdc7-inhi-bition checkpoint arrest, instead accumulating DNA dou-ble strand breaks (�H2AX immunostaining; Figure 5D)while progressing through S phase (increase in the per-centage of cells incorporating BrdU; Figure 5C) to theG2/M boundary, is further supported by the increase incyclin A and cyclin B levels (Figure 5C). The decline inthe G2/M population in doubly depleted Cdc7/p53 cellsand concomitant increase in the number of cells with lessthan 2C DNA content (Figure 5C) suggests that failure tomaintain the Cdc7-inhibition checkpoint leads to apopto-tic cell death. As expected, induction of apoptosis inCdc7/p53 doubly depleted cells was confirmed by theappearance of cells with morphological features of apo-ptosis, positive TUNEL staining of nuclei and procaspase


cleavage (Figure 5D). Thus loss of p53 function in earlybreast multistep tumorigenesis appears to be sufficient tooverride the Cdc7-inhibition checkpoint.

The Cdc7-Inhibition Checkpoint–Mediated CellCycle Arrest Is Fully Reversible in BreastEpithelial Cells

The RNAi data discussed above show that in untrans-formed breast epithelial cells, the Cdc7-inhibition check-

point blocks S phase progression without affecting cellviability. Notably, Cdc7 levels and proliferative capacitywere restored between 96 and 144 hours posttransfec-tion (Figure 6, A and B), most likely due to a wash out ofthe RNAi effect at later time points. An alternative expla-nation for the recovery of cell proliferation several daysposttransfection would be the emergence of a populationof proliferating cells that originated from a small fractionof nontransfected cells. To directly test whether arrestedcells are able to initiate DNA synthesis after recovery ofCdc7 levels, we performed double-labeling of HMEpC

Figure 5. Cell cycle arrest in HMEpC cells following Cdc7 depletion is p53-dependent. A: Immunoblot analysis of WCE prepared from untreated (UT) HMEpCcells and cells transfected with control (CO), CDC7- plus CO- (CO/Cdc7KD), or CDC7- plus p53- (Cdc7KD/p53KD) siRNA probed with antibodies against Cdc7, p53,and actin (loading control). B: At the indicated time points, cell number was measured in UT, CO, CO/Cdc7KD, and Cdc7KD/p53KD cell populations. C: Cell cycleprogression analysis of CO/Cdc7KD and Cdc7KD/p53KD HMEpC cells at the indicated time points, determined by DNA content (left panel), percentage ofCO/Cdc7KD, and Cdc7KD/p53KD HMEpC cells incorporating BrdU (center panel), and WCE immunoblotting with the indicated antibodies (right panel). D:Apoptotic cell death was detected in Cdc7KD/p53KD HMEpC cells but not in CO or CO/Cdc7KD cells by phase-contrast microscopy (left panel), TUNEL staining(center panel), and immunoblotting for procaspase cleavage (right panel).


cells with CldU and IdU30 pre- and posttransfection withCDC7-siRNA according to the protocol shown in Figure6C. Cells were transfected with control- or CDC7-siRNA45 minutes after the addition of CldU. Directly after thetransfection (t0) and 711⁄4 hours (t72) and 1431⁄4hours(t144) posttransfection, siRNA was washed out, and IdUwas added to the medium for the next 45 minutes. Cellswere then fixed and studied by confocal fluorescencemicroscopy with antibodies to CldU (green channel) andIdU (red channel). Representative cells are shown inFigure 6C, revealing different patterns of incorporation ofthe halogenated nucleotides into DNA. Control-trans-fected cells that were exponentially proliferating over thecourse of the experiment continued DNA synthesis at t0which resulted in replication foci that were labeled green,red, or yellow (green � red) in merged images of the twochannels. At t72 and t144, control-transfected cells contin-ued to synthesize DNA as indicated by IdU-incorporation(red pixels), while green pixel intensity per nucleus pro-gressively decreased due to the distribution of incorpo-rated CldU among daughter cells (Figure 6, C and D). InCdc7-depleted cells only a minute population of 5% wereincorporating IdU at t72, whereas the amount of incorpo-rated CldU (green pixel intensity per nucleus) was unaf-fected, consistent with cell cycle arrest. Importantly, in

line with the observed recovery of Cdc7 levels, at t144cells were able to overcome the cell cycle arrest andinitiated DNA synthesis as demonstrated by a 3.5-foldincrease in the percentage of cells with double-labeled(yellow) nuclei (Figure 6, C and D). Thus primary breastepithelial cells respond to loss of Cdc7 function with acheckpoint-induced cell cycle arrest that is fully revers-ible when Cdc7 levels are restored.

Discussion

The systemic treatment for patients with Her2-negativedisease is still limited to endocrine and cytotoxic thera-pies,2 and increasing use of taxanes and anthracyclinesin early stage disease has reduced available therapeuticoptions after relapse. The vertical targeting of growthsignaling pathways, for example with ras and mTOR an-tagonists, has shown efficacy in advanced breast can-cer, but response rates are restricted to around 10% ofpatients.38,39 Signaling pathway redundancy and estab-lishment of autonomous cancer cell cycles potentiallylimits the efficacy of this approach. Novel therapeuticinterventions are therefore urgently required for the treat-

Figure 6. Cell cycle arrest in HMEpC cells after Cdc7 depletion is reversible. A: At the indicated time points, cell number was measured in Cdc7-depleted (Cdc7KD)HMEpC and MCF10A cell populations. B: BrdU incorporation in untreated (UT), control (CO), and CDC7-siRNA transfected HMEpC and MCF10A cells at theindicated time points (relative to CO). C: Cells transfected with CO- or CDC7- (Cdc7KD) siRNA were double-labeled with chlorodeoxyuridine (CldU) andiododeoxyuridine (IdU) as shown (W, washing step). Representative images of individual nuclei for each treatment are shown at high magnification. D: Percentageof nuclei labeled with halogenated nucleotides.


ment of aggressive Her2-overexpressing and triple-neg-ative breast cancers.

Here we have identified Cdc7 kinase as a potentialtherapeutic target in the treatment of Her2-overexpress-ing and triple-receptor negative breast cancers. Cdc7lies at the convergence point of upstream growth signal-ing pathways and may therefore circumvent problemsassociated with pathway redundancy.8 Moreover, Cdc7activity is essential for S phase progression and thereforeremains a potent target in cancer cell cycles that havebecome growth independent. Importantly, dysregulationof Cdc7 in breast cancer appears to be associated withthe development of an aggressive malignant phenotype.Cdc7 overexpression was observed in immortalized andmalignant cell lines when compared with primary mam-mary epithelial cells. Notably, increased Cdc7 expres-sion was found to be linked to arrested tumor differenti-ation, genomic instability, reduced disease-free survival,an accelerated cell cycle phenotype, and Her2 (ER�/PR�/Her2�) and triple-receptor negative (ER�/PR�/Her2�) subtypes, implicating Cdc7 deregulation in thedevelopment of aggressive disease and thus providingfurther target validation for Cdc7 in breast cancer. RNAitargeting of Cdc7 resulted in potent cancer-cell-specifickilling in p53mut triple-negative and Her2-overexpressingbreast cancer cell lines. Primary mammary (HMEpC) andimmortalized nontumorigenic (MCF10A) cell lines, on thecontrary, avoided lethal S phase entry by activating ap53-dependent Cdc7-inhibition checkpoint resulting incell cycle arrest at the G1-S boundary. Notably, RNAitargeting of p53 in HMEpC cells arrested by Cdc7 de-pletion resulted in abrogation of the Cdc7-inhibitioncheckpoint, which was followed by lethal S-G2-M phaseprogression, thus recapitulating the response observedin triple-negative and Her2-overexpressing p53mut can-cer cell lines. Importantly, loss of p53 function is an earlyevent in mammary multistep tumorigenesis, occurring atthe preinvasive stage, with up to 60% of high-gradeductal carcinoma in situ lesions harboring p53 muta-tions.40–43 Moreover, triple-receptor negative and Her2-overexpressing breast cancer subtypes have a high pro-portion, 40–80%, of p53 mutations, in keeping with thefact that these aggressive tumors are thought to arisefrom high-grade ductal carcinoma in situ lesions.16–19,43

These observations indicate that loss of the Cdc7-inhibi-tion checkpoint is likely to occur at an early stage inmammary multistep tumorigenesis and that pharmaco-logical Cdc7 inhibitors currently in development9 maythus have clinical utility not only in the treatment of ag-gressive Her2-overexpressing and triple-receptor nega-tive tumor types but also in the treatment of ductal car-cinoma in situ lesions. In addition, a proportion of luminaltype tumors harbor p53 mutations (9 to 13%) display theaggressive cell cycle phenotype (III) and may thereforerespond to Cdc7 inhibitors.

The double-labeling synthetic nucleoside incorpora-tion experiments clearly demonstrate that the cell cyclearrest after activation of the Cdc7-inhibition checkpoint inprimary somatic cells is reversible on recovery of Cdc7kinase activity, and secondly, that cells remain fully viablewhile arrested at the G1-S boundary. This suggests that

Cdc7 inhibitors may have limited toxicity in self-renewingtissues with high turnover (eg, gut or the hemopoieticsystem), thus widening the potential therapeutic window.Interestingly, most systemic chemotherapeutic regimesfor breast cancer, whether in the adjuvant setting or forthe treatment of metastatic disease, involve combinationsof S phase agents that target DNA synthesis (eg, anthracy-clines and antimetabolites) and M phase agents with anti-mitotic activity (eg, taxanes, vinca alkaloids and epothi-lones).2 The Achilles heel of these chemotherapeuticregimens is that S- and M-phase agents also affect normalcycling cells, resulting in marrow suppression (neutrope-nia), hair loss, and gut toxicity. Activation of the Cdc7-inhibition checkpoint in normal cycling cells using Cdc7inhibitors before systemic chemotherapy with S- and/or M-phase agents may therefore provide a method of shieldingnormal cells from chemotherapeutic agents. In support ofthis concept, we have observed that primary somatic cellsarrested by the Cdc7-inhibition checkpoint are completelyshielded from anti-mitotic cyclotherapy (S. R. Kingsbury, A.Wollenschlaeger, R. Sainsbury, K. Stoeber, G. H. Williams,manuscript submitted for publication).

In summary, our findings show that Cdc7 kinase is apotent and highly specific anti-cancer target in Her2-overexpressing and triple-receptor negative breast can-cers. Pharmacological Cdc7 kinase inhibitors emergingfrom drug development programs worldwide are there-fore likely to significantly broaden the therapeutic arma-mentarium available for treatment of aggressive p53mut

breast cancers, both in the adjuvant and metastatic set-ting. Analysis of cell cycle phenotype, Cdc7 expressionlevels, and p53 status may allow prediction of therapeuticresponse to such inhibitors. Forthcoming clinical trialswith Cdc7 inhibitors will provide an opportunity for thisnovel treatment paradigm to be tested.

Acknowledgments

We thank Joanna Franks and Caroline Richardson forassistance in retrieving archival tissue and recording clin-icopathological parameters.

References

1. Feuer EJ, Wun LM, Boring CC, Flanders WD, Timmel MJ, Tong T: Thelifetime risk of developing breast cancer. J Natl Cancer Inst 1993,85:892–897

2. Miles DW: Recent advances in systemic therapy. When HER2 is notthe target: advances in the treatment of HER2-negative metastaticbreast cancer. Breast Cancer Res 2009, 11:208

3. La Vecchia C, Bosetti C, Lucchini F, Bertuccio P, Negri E, Boyle P,Levi F: Cancer mortality in Europe, 2000–2004, and an overview oftrends since 1975. Ann Oncol 2010, 21:1323–1360

4. Petrelli F, Cabiddu M, Cazzaniga ME, Cremonesi M, Barni S: Tar-geted therapies for the treatment of breast cancer in the post-trastu-zumab era. Oncologist 2008, 13:373–381

5. Di Cosimo S, Baselga J: Targeted therapies in breast cancer: whereare we now? Eur J Cancer 2008, 44:2781–2790

6. Hortobagyi GN: Trastuzumab in the treatment of breast cancer.N Engl J Med 2005, 353:1734–1736

7. Johnston SR: Targeting downstream effectors of epidermal growthfactor receptor/HER2 in breast cancer with either farnesyltransferase


inhibitors or mTOR antagonists. Int J Gynecol Cancer 2006, 16(Suppl2):543–548

8. Williams GH, Stoeber K: Cell cycle markers in clinical oncology. CurrOpin Cell Biol 2007, 19:672–679

9. Swords R, Mahalingam D, O’Dwyer M, Santocanale C, Kelly K, CarewJ, Giles F: Cdc7 kinase - a new target for drug development. Eur JCancer 2010, 46:33–40

10. Machida YJ, Hamlin JL, Dutta A: Right place, right time, and onlyonce: replication initiation in metazoans. Cell 2005, 123:13–24

11. Remus D, Diffley JF: Eukaryotic DNA replication control: lock andload, then fire. Curr Opin Cell Biol 2009, 21:771–777

12. Masai H, You Z, Arai K: Control of DNA replication: regulation andactivation of eukaryotic replicative helicase. MCM IUBMB Life 2005,57:323–335

13. Montagnoli A, Tenca P, Sola F, Carpani D, Brotherton D, Albanese C,Santocanale C: Cdc7 inhibition reveals a p53-dependent replicationcheckpoint that is defective in cancer cells. Cancer Res 2004,64:7110–7116

14. Blow JJ, Gillespie PJ: Replication licensing and cancer–a fatal entan-glement? Nat Rev Cancer 2008, 8:799–806

15. Loddo M, Kingsbury SR, Rashid M, Proctor I, Holt C, Young J,El-Sheikh S, Falzon M, Eward KL, Prevost T, Sainsbury R, Stoeber K,Williams GH: Cell-cycle-phase progression analysis identifies uniquephenotypes of major prognostic and predictive significance in breastcancer. Br J Cancer 2009, 100:959–970

16. Sorlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H, HastieT, Eisen MB, van de Rijn M, Jeffrey SS, Thorsen T, Quist H, MateseJC, Brown PO, Botstein D, Eystein LP, Borresen-Dale AL: Geneexpression patterns of breast carcinomas distinguish tumor sub-classes with clinical implications. Proc Natl Acad Sci USA 2001,98:10869–10874

17. Calza S, Hall P, Auer G, Bjohle J, Klaar S, Kronenwett U, Liu ET, MillerL, Ploner A, Smeds J, Bergh J, Pawitan Y: Intrinsic molecular signa-ture of breast cancer in a population-based cohort of 412 patients.Breast Cancer Res 2006, 8:R34

18. Tan DS, Marchio C, Jones RL, Savage K, Smith IE, Dowsett M,Reis-Filho JS: Triple negative breast cancer: molecular profiling andprognostic impact in adjuvant anthracycline-treated patients. BreastCancer Res Treat 2008, 111:27–44

19. Carey LA, Perou CM, Livasy CA, Dressler LG, Cowan D, Conway K,Karaca G, Troester MA, Tse CK, Edmiston S, Deming SL, Geradts J,Cheang MC, Nielsen TO, Moorman PG, Earp HS, Millikan RC: Race,breast cancer subtypes, and survival in the Carolina Breast CancerStudy. JAMA 2006, 295:2492–2502

20. Bloom HJ, Richardson WW: Histological grading and prognosis inbreast cancer; a study of 1409 cases of which 359 have beenfollowed for 15 years. Br J Cancer 1957, 11:359–377

21. Rampaul RS, Pinder SE, Elston CW, Ellis IO: Prognostic and predic-tive factors in primary breast cancer and their role in patientmanagement: the Nottingham Breast Team. Eur J Surg Oncol 2001,27:229–238

22. Wharton SB, Hibberd S, Eward KL, Crimmins D, Jellinek DA, Levy D,Stoeber K, Williams GH: DNA replication licensing and cell cyclekinetics of oligodendroglial tumours. Br J Cancer 2004, 91:262–269

23. Dudderidge TJ, Stoeber K, Loddo M, Atkinson G, Fanshawe T,Griffiths DF, Williams GH: Mcm2. Geminin, and KI67 define prolifer-ative state and are prognostic markers in renal cell carcinoma. ClinCancer Res 2005, 11:2510–2517

24. Kulkarni AA, Loddo M, Leo E, Rashid M, Eward KL, Fanshawe TR,Butcher J, Frost A, Ledermann JA, Williams GH, Stoeber K: DNAreplication licensing factors and aurora kinases are linked to aneu-ploidy and clinical outcome in epithelial ovarian carcinoma. ClinCancer Res 2007, 13:6153–6161

25. Shetty A, Loddo M, Fanshawe T, Prevost AT, Sainsbury R, WilliamsGH, Stoeber K: DNA replication licensing and cell cycle kinetics ofnormal and neoplastic breast. Br J Cancer 2005, 93:1295–1300

26. Harvey JM, Clark GM, Osborne CK, Allred DC: Estrogen receptorstatus by immunohistochemistry is superior to the ligand-binding

assay for predicting response to adjuvant endocrine therapy in breastcancer. J Clin Oncol 1999, 17:1474–1481

27. Haroske G, Giroud F, Reith A, Bocking A: 1997 ESACP consensusreport on diagnostic DNA image cytometry. Part I: basic consider-ations and recommendations for preparation, measurement and in-terpretation European Society for Analytical Cellular Pathology. AnalCell Pathol 1998, 17:189–200

28. Eward KL, Obermann EC, Shreeram S, Loddo M, Fanshawe T, Wil-liams C, Jung HI, Prevost AT, Blow JJ, Stoeber K, Williams GH: DNAreplication licensing in somatic and germ cells. J Cell Sci 2004,117:5875–5886

29. Kulkarni AA, Kingsbury SR, Tudzarova S, Hong HK, Loddo M, RashidM, Rodriguez-Acebes S, Prevost AT, Ledermann JA, Stoeber K,Williams GH: Cdc7 kinase is a predictor of survival and a noveltherapeutic target in epithelial ovarian carcinoma. Clin Cancer Res2009, 15:2417–2425

30. Seiler JA, Conti C, Syed A, Aladjem MI, Pommier Y: The intra-S-phasecheckpoint affects both DNA replication initiation and elongation: single-cell and -DNA fiber analyses. Mol Cell Biol 2007, 27:5806–5818

31. Kingsbury SR, Loddo M, Fanshawe T, Obermann EC, Prevost AT,Stoeber K, Williams GH: Repression of DNA replication licensing inquiescence is independent of geminin and may define the cell cyclestate of progenitor cells. Exp Cell Res 2005, 309:56–67

32. Neve RM, Chin K, Fridlyand J, Yeh J, Baehner FL, Fevr T, Clark L,Bayani N, Coppe JP, Tong F, Speed T, Spellman PT, DeVries S,Lapuk A, Wang NJ, Kuo WL, Stilwell JL, Pinkel D, Albertson DG,Waldman FM, McCormick F, Dickson RB, Johnson MD, Lippman M,Ethier S, Gazdar A, Gray JW: A collection of breast cancer cell linesfor the study of functionally distinct cancer subtypes. Cancer Cell2006, 10:515–527

33. Mackay A, Tamber N, Fenwick K, Iravani M, Grigoriadis A, Dexter T,Lord CJ, Reis-Filho JS, Ashworth A: A high-resolution integratedanalysis of genetic and expression profiles of breast cancer cell lines.Breast Cancer Res Treat 2009, 118:481–498

34. Montagnoli A, Valsasina B, Brotherton D, Troiani S, Rainoldi S, Tenca P,Molinari A, Santocanale C: Identification of Mcm2 phosphorylation sitesby S-phase-regulating kinases. J Biol Chem 2006, 281:10281–10290

35. Crosio C, Fimia GM, Loury R, Kimura M, Okano Y, Zhou H, Sen S, AllisCD, Sassone-Corsi P: Mitotic phosphorylation of histone H3: spatio-temporal regulation by mammalian Aurora kinases. Mol Cell Biol2002, 22:874–885

36. Connell-Crowley L, Harper JW, Goodrich DW: Cyclin D1/Cdk4 regu-lates retinoblastoma protein-mediated cell cycle arrest by site-spe-cific phosphorylation. Mol Biol Cell 1997, 8:287–301

37. Chi Y, Welcker M, Hizli AA, Posakony JJ, Aebersold R, Clurman BE:Identification of CDK2 substrates in human cell lysates. Genome Biol2008, 9:R149

38. Johnston SR, Hickish T, Ellis P, Houston S, Kelland L, Dowsett M,Salter J, Michiels B, Perez-Ruixo JJ, Palmer P, Howes A: Phase IIstudy of the efficacy and tolerability of two dosing regimens of thefarnesyl transferase inhibitor. R115777, in advanced breast cancerJ Clin Oncol 2003, 21:2492–2499

39. Chan S, Scheulen ME, Johnston S, Mross K, Cardoso F, Dittrich C,Eiermann W, Hess D, Morant R, Semiglazov V, Borner M, Salzberg M,Ostapenko V, Illiger HJ, Behringer D, Bardy-Bouxin N, Boni J, Kong S,Cincotta M, Moore L: Phase II study of temsirolimus (CCI-779), a novelinhibitor of mTOR, in heavily pretreated patients with locally advanced ormetastatic breast cancer. J Clin Oncol 2005, 23:5314–5322

40. Arpino G, Laucirica R, Elledge RM: Premalignant and in situ breastdisease: biology and clinical implications. Ann Intern Med 2005,143:446–457

41. Nofech-Mozes S, Spayne J, Rakovitch E, Hanna W: Prognostic andpredictive molecular markers in DCIS: a review. Adv Anat Pathol2005, 12:256–264

42. Meijnen P, Peterse JL, Antonini N, Rutgers EJ, van de Vijver MJ:Immunohistochemical categorisation of ductal carcinoma in situ ofthe breast. Br J Cancer 2008, 98:137–142

43. Wiechmann L, Kuerer HM: The molecular journey from ductal carcinomain situ to invasive breast cancer. Cancer 2008, 112:2130–2142


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Targeting DNA Replication before it Starts